Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Normal and Ha-ras-1 oncogene transformed Buffalo rat liver (BRL) cells show differential resistance to cytoskeletal protein inhibitors.

In the present study, using immunofluorescence microscopy, we have demonstrated that normal and Ha-ras-1 transformed Buffalo rat liver (BRL) cells which were exposed to cytoskeletal protein inhibitors, showed a differential resistance of their microfilament and microtubule networks. One hour exposure of normal BRL cells to 10(-5) M cytochalasin B provoked a clear and already total breakdown of actin filaments. However, at this concentration of cytochalasin B, the microfilaments of transformed BRLHO6T1-1 cells were not seriously affected; a higher cytochalasin B concentration (> or = 2 x 10(-5) M) was required to induce a significant breakdown of microfilaments in these transformed cells. The two cell lines also demonstrated differential microtubule stability when they were treated with either colchicine or triethyllead. Three hours exposure to 10(-6) M of either antimicrotubule agents was sufficient to disrupt the microtubules of normal BRL cells, without affecting their counterparts in the transformed BRLHO6T1-1 cells. A 10-fold higher drug concentration (10(-5) M) was required to induce microtubular breakdown in the transformed BRL cells. The differential stability of microfilaments and microtubules in normal and transformed BRL cells that was observed could not be attributed to a differential internalization of the agents, as shown by experiments on the uptake of [3H]-cytochalasin B and triethyllead. In addition, the transformed BRLHO6T1-1 cells did not express altered actin and tubulin isoforms, as demonstrated by isoelectric focusing followed by immunoblotting analysis. We conclude that the transformation of BRL cells with the Ha-ras-1 oncogene results in a greater stability of microfilaments and microtubules, leading to a structurally firmer cell shape.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colchicine binding in the free-living nematode Caenorhabditis elegans

The [3H]colchicine-binding activity of a crude supernatant of the free-living nematode Caenorhabditis elegans was resolved into a non-saturable component and a tubulin-specific component after partial purification of tubulin by polylysine affinity chromatography. The two fractions displayed opposing thermal dependencies of [3H]colchicine binding, with non-saturable binding increasing, and tubulin binding decreasing, at 4 degrees C. Binding of [3H]colchicine to C.elegans tubulin at 37 degrees C is a pseudo-first-order rate process with a long equilibration time. The affinity of C. elegans tubulin for [3H]colchicine is relatively low (Ka = 1.7 x 10(5) M(-1)) and is characteristic of the colchicine binding affinities observed for tubulins derived from parasitic nematodes. [3H]Colchicine binding to C. elegans tubulin was inhibited by unlabelled colchicine, podophyllotoxin and mebendazole, and was enhanced by vinblastine. The inhibition of [3H]colchicine binding by mebendazole was 10-fold greater for C. elegans tubulin than for ovine brain tubulin. The inhibition of [3H]colchicine binding to C. elegans tubulin by mebendazole is consistent with the recognised anthelmintic action of the benzimidazole carbamates. These data indicate that C. elegans is a useful model for examining the interactions between microtubule inhibitors and the colchicine binding site of nematode tubulin.     

The glucose transport system of muscle plasma membranes: characterization by means of [3H]cytochalasin B binding

A membrane-rich preparation was isolated from adult rat skeletal muscle in low salt media and further fractionated in sucrose gradients. Fraction F2, with a relative density of 1.092-1.119, consisted of sealed membrane vesicles which were enriched in plasma membrane markers. These vesicles were capable of stereospecific D-glucose uptake which was sensitive to cytochalasin B (CB). The membranes were also enriched in high affinity [3H]CB binding activity (Kd of 0.28 microM). [3H]CB binding to the glucose carrier of these plasma membranes, estimated as the fraction of binding protectable by D-glucose, ranged between 2.5 and 7.4 pmol/mg protein in several membrane preparations. The amount of [3H]CB binding to muscle membranes from newborn and adult rats was not markedly different. Trypsin, at low concentrations, altered the molecular weight of several membrane components, without affecting [3H]CB binding. Higher concentrations of trypsin abolished [3H]CB binding. Both 2,4-dinitrofluorobenzene (0.1 mM) and N-ethylmaleimide (15 mM) inhibited [3H]CB binding; inhibition by these reagents was prevented by inclusion of micromolar concentrations of CB in the reaction mixture. Several procedures that extracted specific proteins enriched the D-glucose-sensitive [3H]CB binding to the protein-depleted membranes. Antibody raised against the glucose carrier of human red cell membranes cross-reacted with a polypeptide of Mr about 45K of muscle membranes which might represent the glucose carrier.     

Role of membrane colchicine binding proteins in the transmission of prolactin message to casein genes in the rabbit mammary gland

Previous work demonstrated that tubulin binding drugs specifically inhibit the capacity of prolactin to initiate casein and DNA synthesis in the mammary cell. It was concluded that microtubules or other tubulin containing cellular structures were involved in the transmission of the prolactin message to genes. In the present work, it is shown that griseofulvin, an antimitotic drug which alters microtubule structure and function, does not prevent prolactin actions. Autoradiographic studies showed that [3H]colchicine binds preferentially to plasma and Golgi membranes in the mammary cell. Short term cultures of mammary explants with [3H]colchicine demonstrated that the labelled drug binds to membranous cellular structures which were isolated from explants at the end of the culture. Fractions containing plasma and Golgi membranes contained the highest amount of radioactivity. Solubilisation of the membranes by Triton X-100 dissociated the [3H]colchicine from the prolactin receptors as judged by a chromatography of the soluble fraction on a Sepharose 6 B column. On the column, the labelled colchicine remains associated with a molecular entity which may be free tubulin. In all cases, the binding of [3H]colchicine was greatly attenuated by an excess of unlabelled colchicine but was only slightly affected by the competition with lumicolchicine. These results suggest that mammary membranes contain tubulin and that binding of drugs to this molecule inhibits the generation of the prolactin second messengers eliciting the hormonal actions in the mammary cell. This also suggests that microtubules are probably not involved in the mechanism of prolactin action.     

Proteolytic cleavage of [3H]nitrobenzylthioinosine-labelled nucleoside transporter in human erythrocytes.

The transmembrane topology of the nucleoside transporter of human erythrocytes, which had been covalently photolabelled with [3H]nitrobenzylthioinosine, was investigated by monitoring the effect of proteinases applied to intact erythrocytes and unsealed membrane preparations. Treatment of unsealed membranes with low concentrations of trypsin and chymotrypsin at 1 degree C cleaved the nucleoside transporter, a band 4.5 polypeptide, apparent Mr 66 000-45 000, to yield two radioactive fragments with apparent Mr 38 000 and 23 000. The fragment of Mr 38 000, in contrast to the Mr 23 000 fragment, migrated as a broad peak (apparent Mr 45 000-31 000) suggesting that carbohydrate was probably attached to this fragment. Similar treatment of intact cells under iso-osmotic saline conditions at 1 degree C had no effect on the apparent Mr of the [3H]nitrobenzylthioinosine-labelled band 4.5, suggesting that at least one of the trypsin cleavage sites resulting in the apparent Mr fragments of 38 000 and 23 000 is located at the cytoplasmic surface. However, at low ionic strengths the extracellular region of the nucleoside transporter is susceptible to trypsin proteolysis, indicating that the transporter is a transmembrane protein. In contrast, the extracellular region of the [3H]cytochalasin B-labelled glucose carrier, another band 4.5 polypeptide, was resistant to trypsin digestion. Proteolysis of the glucose transporter at the cytoplasmic surface generated a radiolabelled fragment of Mr 19 000 which was distinct from the Mr 23 000 fragment radiolabelled with [3H]nitrobenzylthioinosine. The affinity for the reversible binding of [3H]cytochalasin B and [3H]nitrobenzylthioinosine to the glucose and nucleoside transporters, respectively, was lowered 2-3-fold following trypsin treatment of unsealed membranes, but the maximum number of inhibitor binding sites was unaffected despite the cleavage of band 4.5 to lower-Mr fragments.     

Binding of [3H]forskolin to solubilized preparations of adenylate cyclase

The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS).     

Effects of four agents that modify microtubules and microfilaments (vinblastine,colchicine, lidocaine,and cytochalasin B) on macrophage-mediated cytotoxicity to tumor cells

Summary The effects of vinblastine, colchicine, lidocaine, and cytochalasin B on tumor cell killing by BCG-activated macrophages were examined. These four drugs were selected for their action on membrane-associated cytoskeletal components, microtubules, and microfilaments. Colchicine and vinblastine, which block microtubular synthesis, inhibit macrophage-mediated tumor-cell cytotoxicity at a concentration of 10^–6 M. Cytochalasin B, which disrupts microfilaments, enhances tumor cell lysis and stasis due to activated macrophages at a concentration of 10^–7 M. Lidocaine, which may induce the disappearance of both microtubules and microfilaments, has the same inhibiting effect as vinblastine at a concentration of 5×10^–7 M. Whereas vinblastine and lidocaine seem to act on the macrophage itself, cytochalasin B exerts its effect predominantly on the tumor cell. These results suggest that microtubules and microfilaments play a role in the destruction of tumor cells by activated macrophages.     

Binding of [3H]cytochalasin B to tumoral islet cells

Tumoral pancreatic islet cells of the RINm5F line are equipped with two classes of [3H]cytochalasin B binding sites with respective Kd of 0.4 and 7 microM. The binding of the fungal metabolite and its dissociation from the binding sites display rapid time courses. The binding is inhibited by D-glucose, more than by L-glucose, by phlorizin and by cytochalasin E. These findings are considered in the light of the dual action of cytochalasin B upon hexose transport and motile activity in islet cells.     

Biochemical characterization of [3H]tryptamine binding sites from rat brain

Rat brain membranes were treated with different protein modifying reagents, all of which were able to reduce [3H]tryptamine binding. However, inactivation by N-ethylmaleimide and iodoacetamide only was counteracted by coincubation with tryptamine. Thus, the [3H]tryptamine binding molecule is a membrane protein with an essential sulfhydryl group at the binding site. After incubation of digitonin-solubilized membranes with seven different lectins, no precipitation of [3H]tryptamine binding sites was observed. On concanavalin A and wheat germ agglutinin affinity chromatography, no [3H]tryptamine binding activity was found to be specifically bound. Therefore, the [3H]tryptamine binding protein appears to be devoid of lectin binding carbohydrate residues.     

Association of actin with chromaffin granule membranes and the effect of cytochalasin B on the polarity of actin filament elongation

Membranes of chromaffin granules isolated from bovine adrenal medulla are shown to bind dihydrocytochalasin B with high affinity. These membranes also bound [3H]actin in a time- and Mg2+-dependent manner and electron microscopy showed the presence of membrane-attached actin filaments following addition of exogenous actin. Binding of [3H]actin was partially inhibited by cytochalasin B. Electron microscopic analysis of heavy meromyosin-decorated, membrane-attached filaments showed terminally (end-on) attached filaments with both possible polarities (i.e., filaments with arrowheads pointing both towards and away from the membranes). Treatment of samples with cytochalasin B preferentially inhibited growth of filaments with their 'barbed' ends pointing away from membranes. These results are discussed with respect to the role of actin in secretory granule function and the mechanism of cytochalasin action.     

Effects of trypsin on binding of [3H]epinephrine and [3H]-dihydroergocryptine to rat liver plasma membranes. Evidence for interconversion of binding sites

Treatment of liver plasma membranes with trypsin at low concentrations (1 to 2 microgram/mg of protein) caused at 3- to 4-fold increase in alpha-specific [3H]epinephrine binding. The change was due to an increase in the number of high affinity binding sites, with no change in the dissociation constant. With increasing trypsin concentrations, the dissociation constant was decreased and there was a progressive loss of binding. Elastase, papain, and thermolysin caused similar effects, whereas the thrombin, leucine aminopeptidase, phospholipase A2, phospholipase C, phospholipase D, and detergents did not cause an increase in [EH]epinephrine binding. The increase in epinephrine high affinity binding sites was correlated with a loss of high affinity [3H]-dihydroergocryptine binding sites which also bind [3H]epinephrine with low affinity (El-Refai, M. F., Blackmore, P. F., and Exton, J. H. (1979) J. Biol. Chem. 254, 4375-4386). Incubation of membranes with the alpha blockers dihydroergocryptine (50 nM) and phenoxybenzamine (20 nM) prior to protease treatment diminished the increase in [3H]epinephrine binding induced by trypsin (1.5 microgram/mg). The concentration dependence and time course of trypsin actions on 70 nM [3H]epinephrine binding and 10 nM [3H]dihydroergocryptine binding are consistent with a trypsin-mediated conversion of low affinity epinephrine binding sites to high affinity epinephrine binding sites.     

Implication of microtubules and microfilaments in the response of the ovarian adenylate cyclase-cyclic AMP system to gonadotropins and prostaglandin E2.

Addition of anti-actin serum or cytochalasin B (3 μg/ml) to the medium abolished the stimulatory effect of LH and of choleragen, and inhibited the action of FSH, but not of PGE₂, on cyclic AMP production in cultured rat Graafian follicles. Colchicine and anti-sera to BSA, tubulin or smooth-muscle myosin, as well as anti-actin serum absorbed with actin, had no effect on the follicular response to LH, but anti-tubulin serum and colchicine inhibited the response to FSH and PGE₂. The inhibitory effect of cytochalasin B on LH-action was fully reversed 24 h after transfer of the follicles to drug-free medium. Neither anti-actin serum nor cytochalasin B had any effect on the binding of ¹²⁵I-hCG by the follicular cell membrane. The results suggest that microfilaments, but not microtubules, are intimately involved in the process of LH- and choleragen-stimulated ovarian adenylate cyclase activity. By contrast, the action of PGE₂ is dependent on microtubule assembly, while the action of FSH seems to depend on both these components of the cytoskeleton.     

Characterization of alpha-adrenoceptor subtypes by [3H]prazosin and [3H]rauwolscine binding to canine venous smooth muscle membranes

Postsynaptic alpha-adrenoceptor subtypes were studied using [3H]prazosin and [3H]rauwolscine binding to plasmalemma-enriched microsomal fractions isolated from dog saphenous veins and mesenteric veins. Both radioligands showed saturable binding consistent with the presence of a single homogeneous binding site in each case, based on Scatchard analysis. The Kd values of [3H]prazosin and [3H]rauwolscine, calculated from kinetic studies were similar to those from equilibrium binding data in both venous muscle membranes. The microsomal membranes of dog saphenous vein and mesenteric vein contained about a fourfold higher density of the high affinity [3H]rauwolscine binding sites than those for [3H]prazosin binding. In competition studies, IC50 values for displacement of rauwolscine or prazosin suggested that the sites of interaction for the antagonists prazosin and rauwolscine were independent. Phenylephrine, a functionally selective alpha-adrenoceptor agonist, competed with a similar IC50 value for the specific binding sites of [3H]prazosin and [3H]rauwolscine; but B-HT 920, a functionally selective alpha 2-adrenoceptor agonist, competed for [3H]rauwolscine and [3H]prazosin binding with distinctly different IC50 values. Our data show the existence of two populations of alpha-adrenoceptor antagonist binding sites in the plasma membranes of dog saphenous vein and mesenteric vein, and raise the question whether agonist selectively depends on different affinities or on differential efficacies at one or two sites.     

Differential interactions of muscarinic drugs with binding sites of [3H]pirenzepine and [3H]quinuclidinyl benzilate in rat brain tissue

Some atypical muscarinic drugs were compared with classical drugs with respect to inhibition of specific binding of [3H]pirenzepine ([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB) to membrane preparations of rat brain. The interactions of the agonists McN-A343 and carbachol with [3H]QNB at muscarinic sites in brain stem preparations were differently modulated in the presence of an excess of PZ. Moreover, McN-A343 exhibited a preferential affinity for [3H]PZ sites in whole brain membranes whereas carbachol bound with high affinity to [3H]QNB sites in brain stem preparations. Various muscarinic agonists and antagonists displayed different affinity patterns in the [3H]PZ and [3H]QNB binding. These data are indicative of two populations of pharmacologically distinguishable binding sites and support the concept of muscarinic receptor heterogeneity in rat brain.     

Comparison of [3H] phencyclidine ([3H] PCP) and [3H] N-[1-(2-thienyl) cyclohexyl] piperidine ([3H] TCP) binding properties to rat and human brain membranes

The investigation of [3H] PCP and [3H] TCP binding properties to rat cerebrum and cerebellum resulted in the demonstration of multiple binding sites for the two drugs. In the two tissue preparations PCP had a lower affinity than TCP. In membranes from the cerebrum an equal number of high affinity binding sites were present for [3H] PCP and [3H] TCP. However, low affinity binding sites were two times more numerous for [3H] PCP than for [3H] TCP. In the cerebellum, the number of high and low affinity sites labeled by the two radioligands was identical, but the number of high affinity sites was about 7 fold lower than in the cerebrum. Taken together these results may indicate that in the cerebrum [3H] PCP labels other sites than NMDA/PCP receptor(s), maybe sigma receptors and/or the dopamine uptake complex. In human cerebral cortex samples [3H] TCP also bound to two different sites. The number of high and low affinity sites were 12 and 3 times, respectively, less abundant than in the rat cerebrum. Low affinity sites were of higher affinity (5 times) than corresponding sites in the rat brain. In the human cerebellum [3H] TCP binding parameters were identical to those measured in the same region in the rat.     

Interaction of [3H]AMP with liver cells and their plasma membranes

Specific binding of [3H]AMP to rat hepatocytes and their plasma membranes was studied. It was shown that the time course of this binding reached a maximum within the first 15 seconds. An equilibrium binding study revealed the presence of a single class of binding sites with Kd of 20 microM both in hepatocytes and in plasma membranes. The [3H]AMP binding sites were inactivated by treatment with trypsin as well as by heating. 5'-Phosphorylated derivatives of adenosine (ATP, ADP) effectively competed with [3H]AMP for the binding sites, while adenosine, beta-glycerophosphate and 3'-AMP were inactive. The binding of [3H]AMP increased by 400% in the presence of concanavalin A, a specific inhibitor of plasma membrane 5'-nucleotidase. It was concluded that the catalytic center of 5'-nucleotidase is a receptor for adenine nucleotides.     

5'-N-ethylcarboxamido [3H] adenosine binding sites of mouse P815 mastocytoma cell membranes: solubilization and partial purification by affinity chromatography

A 5'-N-ethylcarboxamido[3H]adenosine ([3H]NECA) binding site of mouse mastocytoma P815 cell membranes has been purified approximately 100-fold by affinity chromatography. This adenosine binding site, which has a similar specificity to that of the A2 adenosine receptor, was absorbed on NECA-linked Sepharose 6B and eluted with NECA. The adsorption of the [3H]NECA binding site to the affinity matrix was specifically blocked by NECA. The [3H]NECA binding site bound on the affinity matrix was also specifically eluted by NECA. This affinity matrix adsorbed approximately 90% of the digitonin-solubilized [3H]NECA binding activity applied, and after the gel was washed, 30-50% of the adsorbed binding activity could be eluted with 500 microM NECA with specific binding activity of 50-70 pmol/mg of protein. The affinity-purified [3H]NECA binding site retained the same ligand binding specificities as the original membrane preparation. The results indicate that the NECA-Sepharose Sepharose 6B should provide a powerful tool for the eventual purification of [3H]NECA binding sites of P815 cell membranes.     

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Effect of colchicine on the redistribution of L-[1,5,6-3H]fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes.

To define the role of cytoplasmic microtubules in the biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells, we studied the effect of colchicine on the incorporation of L-[1,5,6-3H]fucose into Golgi, lateral basal and microvillus membranes. Colchicine was administered intraperitoneally before or after injection of radioactive fucose. The incorporation of radioactivity into Golgi membranes was little affected by colchicine, which did not prevent the redistribution of most of the labelled glycoproteins from the Golgi complex into other parts of the villus cell. The incorporation of labelled glycoproteins into the microvillus membrane was greatly inhibited by colchicine given 2 h or 10 min before the radioactive fucose: all labelled glycoproteins present in this membrane were equally affected. In contrast, the administration of colchicine considerably increased the incorporation of radioactivity into the lateral basal part of the plasmalemma, and prevented the disappearance of most of the labelled glycoproteins from this membrane at late times after fucose injection. These results suggest that cytoplasmic microtubular structures are important for the polarization of the intestinal villus cell and the biogenesis of the microvillus membrane, although playing little or no role in the movement of membrane components from the Golgi complex to the lateral basal part of the plasmalemma.     

Glutaraldehyde pretreatment blocks temperature-induced high-affinity [3H]tryptamine binding

The effect of glutaraldehyde (and Azure A) on temperature-sensitive high-affinity [3H]tryptamine binding was investigated in rat brain synaptic plasma membranes. In the 0.01-0.1% concentration range, the glutaraldehyde pretreatment preferentially inhibited only the above-mentioned portion of the binding, whereas the posttreatment of this reagent had no effect. On the other hand, in cases of pretreatment or posttreatment, a concentration of glutaraldehyde as high as 0.1% was inactive on the basal [3H]ligand binding capacity of the membranes (i.e., temperature-independent binding). Furthermore, it was revealed that the Scatchard plot of [3H]tryptamine binding in membranes pretreated with glutaraldehyde (0.05%) conformed to a straight line, as did a similar plot of temperature-independent binding. And, it was interesting to find that the binding parameters (KD and Bmax values) of both samples corresponded closely to each other. On the contrary, in all concentrations, Azure A affected nonspecifically both the temperature-dependent and the independent [3H]tryptamine binding to the same degree, regardless of whether or not there was pretreatment or posttreatment. All these observations clearly demonstrate that an appropriate concentration (0.01-0.1%) of glutaraldehyde pretreatment specifically blocks the temperature-induced allosteric modifications of high-affinity [3H]tryptamine binding sites.